Beta-adrenergic receptors (BARs) are members of the G protein-coupled receptor (GPCR) superfamily and couple to the stimulatory G protein (Gs) to activate adenylyl cyclase in response to epinephrine and norepinephrine. We have been investigating the regulation of this signaling pathway and focusing on differences in regulation of the three beta-subtypes, which in order of susceptibility rank as B2AR> B1AR >>B3AR. This may provide a physiological basis for the existence of three subtypes, especially in cells and tissues such as adipose, cardiac and neural that express more than one subtype. Previously, we showed that B1AR undergoes less agonist- stimulated phosphorylation, ubiquitination, desensitization, internalization and down-regulation than B2AR. Phosphorylation of the C-tail of B2AR by GPCR kinases (GRKs) is followed by arrestin binding which uncouples the receptor from Gs resulting in desensitization. In addition, arrestin binds clathrin and AP-2 and promotes receptor internalization by the clathrin coated pit pathway. While B1AR endocytosis is by the same pathway, the two subtypes are sorted to different endosomal compartments. Arrestin also recruits ubiquitin ligases that conjugate ubiquitin to lysine residues of the B2AR. In contrast, B1AR is not ubiquitinated. Upon removal of agonist, both subtypes recycle at similar rates back to the plasma membrane. Upon prolonged agonist treatment, B2AR is degraded and down-regulated more than B1AR. Using chimeras in which the C-tails have been exchanged, we demonstrated that the C-tail is the major determinant of subtype differences in these regulatory processes. A major area of interest in GPCR research are the mechanisms of intracellular sorting of receptors between recycling and degradation pathways. Current models envision sorting motifs on the receptor that are recognized by specific sorting proteins. Both phosphorylation and ubiquitination are prime candidates for receptor motifs. Based on our finding that ubiquitination of B2AR is dependent on its C-tail, we replaced the 3 C-tail lysines with arginines (K348/372/ 375R) to generate the mutant 3K/R. 3K/R underwent less agonist-mediated ubiquitination than wild type (WT) B2AR. Degradation and down-regulation were reduced in the mutant whereas internalization and recycling were similar to WT. Intracellular trafficking of 3K/R was more similar to B1AR than B2AR and the mutant did not accumulate in lysosomes as did WT. Agonist-stimulated phosphorylation of both was similar but upon agonist removal, dephosphorylation of 3K/R occurred 3-4 times more rapidly than WT. As the increased rate was observed during cell-free dephosphorylation, it appears to be intrinsic to the mutation. Okadaic acid, a potent phosphatase inhibitor, reduced the dephosphorylation of WT and 3K/R in both cell-based and cell-free assays. In okadaic acid-treated cells, trafficking of 3K/R to lysosomes increased as did receptor degradation. Our findings indicate that both phosphorylation and ubiquitination are involved in the intracellular sorting of B2AR between recycling to the plasma membrane and lysosomal degradation. Phosphorylation alone appears to be a sufficient signal for degradation whereas ubiquitination may enhance the signal as well as protect the receptor from dephosphorylation.